The subject invention is in the field of immunoassays for determining, an analyte, especially an analyte adsorbed at a surface, or analyte as it occurs in a liquid sample in solution or at a particular location therein, such as at a specific surface.
More in particular, the invention relates to enzymelinked immunosorbance assays (ELISA""s) for determining the concentration of an analyte in a liquid sample, including its concentration in the bulk of the liquid sample or its concentration at a particular surface. Specifically, the present invention relates to new immunoassay methods and kits specifically adapted for carrying out these new immunoassay methods.
Immunoassays, such as ELISA""s, are widely used for the determination, either qualitative or, mostly, quantitative, of a nearly unlimited variety of organic substances, either of natural origin or synthetic chemical compounds, such as peptides, proteins, enzymes, hormones, vitamins, drugs, carbohydrates, etc., for various purposes, such as in particular for diagnostic purposes, but also for forensic applications, food quality control, and generally for any analytic purpose. All such substances to be assayed will be referred to herein generally as analytes.
Many different variants of ELISA methods exist. The description given hereunder aims at illustrating a typical ELISA technique. It does not pretend to be complete and should not be construed in any way as restricting the scope of the present invention. For example, in the following description, ELISA methods are described as comprising separate steps of incubating a sample with a first binding partner of the analyte and incubating the reaction product formed with a second binding partner of the analyte (herein binding partners of the analyte will sometimes be referred to as binding partners for the analyte). However, some existing ELISA embodiments do not comprise such separate incubation steps and allow the analyte to react simultaneously, or shortly one after the other, in one and the same incubation step, with both its first and second binding partners. Competitive ELISA""s are another example of ELISA variants not discussed in detail herein. The subject invention is in principle applicable to any and all ELISA variants, and to similar immunoassay methods which, strictly speaking, are not ELISA methods, e.g. because they do not involve the use of an enzyme.
In a typical ELISA, to detect the presence, or measure the concentration, of an analyte of interest, especially in a liquid sample, which may be a body fluid such as blood, plasma, serum, urine, saliva, sputum, etc., the sample is contacted with a first binding partner for the analyte, and the sample and the binding partner for the analyte are incubated for a sufficient time to allow analyte contained in the sample to bind to the binding partner.
A typical example of such a binding partner is an analyte-specific antibody, e.g. a monoclonal antibody with specificity for the analyte in question. However, other kinds of substances and structures may also qualify as a binding partner. For example, the natural receptor of the analyte in question could be useful as a binding partner, as well as other substances and structures to which the analyte can bind. The binding partner would normally be a specific binding partner, but this is not a requirement. It is even possible to work without a first binding partner and to immobilize (i.e. bind to a solid phase) the analyte either directly (e.g. by adsorption) or through a non-specifically binding linker substance. All of such variants are explicitly intended to be included in the scope of this invention. The words xe2x80x9canalyte adsorbed at a surfacexe2x80x9d as used herein refer to any method resulting in attachment or binding of analyte to a surface.
Usually, said first binding partner is used in an immobilized form, i.e. attached to a solid phase, such as polystyrene beads or the inner surface of the reaction container (e.g. a reaction tube or a well of a microtiter plate). The binding partner may be physically adsorbed onto the solid phase or, usually, be attached by covalent binding. According to some ELISA embodiments, the binding partner is attached by using a suitable coupling agent, and in others by using appropriate linker substances, such as biotin and (strept)avidin. In some ELISA embodiments, the immobilization of the binding partner is carried out after the incubation of the sample and binding partner, thereby allowing the reaction between analyte and binding partner to proceed in the liquid phase. To allow its subsequent immobilization, said binding partner may be applied in a biotinylated form. Immobilization can then be effected by using a solid phase carrying (strept)avidin.
As a result of this first reaction, any analyte present in the sample will have become bound to its binding partner and thereby to the solid phase. Usually after the liquid has been removed and the solid phase has been washed, steps are taken to make the result detectable. The solid phase having attached thereto the binding partner and analyte, if any, is contacted with a second binding partner for the analyte. Again, a specific binding partner is the rule, but not strictly required. Usually, this second binding partner carries a label/marker allowing its detection. In some ELISA embodiments, however, the second binding partner is used in unlabelled form and is labelled after its binding by using a labelled binding partner for the second binding partner. As an example thereof, the second binding partner may be a mouse antibody (either polyclonal or monoclonal) against the analyte in question, and after its binding to the analyte which had been attached via its first binding partner to the solid phase, a labelled goat anti-mouse IgG is used to attach a label to the immobilized complex.
In ELISA""S, the label consists of an enzyme capable of a detectable conversion of a substrate, e.g. a peroxidase such as horseradish peroxidase, capable of converting, in the presence of hydrogen peroxide, a substrate, such as 3,3xe2x80x25,5xe2x80x2-tetramethylbenzidine, into a coloured product.
Normally, after the enzyme-labelled reactant has been attached to the immobilized complex, the solid phase with complex bound thereto is washed before the actual detection phase is entered.
In the detection phase, substrate solution is added to the solid phase with attached complex and the conversion, if any, of the substrate is detected. To allow quantitative measurement of the analyte, the solid phase is incubated with the substrate solution for a fixed time, which should be sufficiently long to allow a substantial enzymatic conversion of the substrate into a coloured substance. After termination of the substrate-converting reaction the intensity of the colouration, which is proportional to the immobilized amount of enzyme, is measured by optical means, such as a photometer to measure the absorbance at a chosen wavelength, such as 450 nm.
A disadvantage of the existing ELISA techniques is that the adsorption and detection phases, to secure assay sensitivity, are very time consuming for low analyte concentrations. The rate of adsorption of analyte to the surface is proportional to the concentration of analyte in the solution, and thus will also become very low, even in well-stirred systems. To allow a reliable measurement of analytes present in a liquid at a concentration in the order of nanograms or even picograms per ml, the adsorption phase may require a reaction time of one to several hours.
Another disadvantage of the existing ELISA techniques is that they do not allow to measure extremely low surface concentrations of analytes, such as occur at the surface of biological (model) membranes.
An object of this invention is to provide a modified immunoassay, e.g. ELISA, technique allowing to reduce the incubation time in the adsorption and/or detection phase, or to increase the sensitivity of the assay, or both.
A further object of this invention is to provide a modified immunoassay, e.g. ELISA, technique which allows to measure analyte concentrations at a particular surface.
Another object of this invention is to provide products specifically adapted for carrying out such modified immunoassay techniques.
The invention pertains to a fundamental modification of the assay principle of immunoassays, e.g. ELISA""s, in which the measurement of the accumulation of enzyme product in the (bulk phase) solution is replaced by in-situ measurement of the accumulation of precipitate on a solid surface. The subject invention involves the use of a precipitate forming system, such as an enzyme-substrate combination which leads to the formation of a precipitate on a solid surface, and measurement of the surface mass, e.g. by ellipsometry or other surface mass measurement techniques.
By using this invention, very low concentrations of adsorbed analyte on the solid surface can be determined and the adsorption phase of analyte from the liquid to the surface can be substantially shortened. Also, the time-consuming build up of product concentration in the bulk liquid phase is no longer required, and is replaced by an adsorption of a thin layer of precipitate, of only molecular dimensions, on the solid surface. This modification implies a large increase in assay sensitivity and offers the possibility of much shorter assay times. It also allows to determine extremely low surface concentrations as may occur at the surface of biological (model) membranes.
Determination of analyte concentrations by xe2x80x98immunoprecipitationxe2x80x99 is a well known technique. The precipitate of analyte-antibody aggregates, formed by this method after addition of the precipitating antibody, is usually simply allowed to sediment, collected and weighed. In spite-of poor precipitation when either the analyte or the antibody is present in large excess, it has been shown that under well defined conditions this technique allows quantitative interpretation of data (see e.g. European patent application 0 368 462). In a more sensitive and rapid version of this technique, (latex) particles coated with antibody are added and aggregate formation is measured by light scattering. In this way, the time-consuming sedimentation is avoided and measurements may be completed in seconds. The present method is fundamentally different from these techniques. In said techniques the analyte is not concentrated at a solid/liquid interface and the amount of precipitate is not continually increasing in time, as when it is produced by an enzyme. Due to these factors, and due to a specific aggregation, such techniques have a limited sensitivity and generally do not allow measurement of analyte concentrations below the ng/ml range.
Analytical methods which involve an enzyme-directed formation of a (coloured) precipitate are known per se. Such a method is known for example in the field of immunocytochemistry. In this known method, to demonstrate the presence and/or localization of various tissue proteins in histological studies, a specific protein in the tissue is determined by first producing thin tissue sections, after freezing the tissue or using various forms of chemical fixation, and then adding antibodies which specifically recognize and bind to the protein. Excess antibodies are removed and then a second, enzyme-linked, antibody, for instance an IgG-HRP conjugate, is added. After removal of excess conjugate, a suitable substrate, such as 3,3xe2x80x2-diaminobenzidine (DAB), is added. If the specific protein is present in the tissue, a localized staining due to the formation of a DAB-derived precipitate will occur.
The present invention, however, does not relate to the localization and detection of proteins in tissue but to the detection and measurement of analyte (concentrations) adsorbed at solid surfaces or present in solutions. Furthermore, the subject invention applies a (preferably quantitative) measurement of surface mass (in the ng/cm2 range) whereas, in said known immunocytochemical method, qualitative results are obtained, usually by visual or. microscopic inspection. In some studies, the intensity of staining has been measured by applying optical density measurements, but the technique has never been combined with a technique for quantitative measurement of surface mass, such as ellipsometry or others. This would also be very difficult to perform because the thickness of tissue sections (micrometers) is of another order of magnitude than the thickness of the precipitate layers (nanometers) measured in the method of the present invention.
The prior art suggests to use immunoassays based on optical interference on specially prepared optically active receptive surfaces, coated by thin oxide-, nitride or polymer films. Using such pre-coating, the additional adsorption of a precipitate layer results in visually detectable colour changes. Such applications have been presented in the U.S. Pat. No. 5,418,136. Apart from visual detection of precipitate formation, measurement of the precipitate by ellipsometry was also applied. However, the various rinsing and/or drying steps required in these techniques preclude the high sensitivity that can be obtained by in-situ measurement. For the detection of very low analyte concentrations in. biological fluids like plasma, serum, blood, milk or urine, the a specifically adsorbed mass of other bulk substances, such as albumin, will often exceed the minute amounts of adsorbed precipitate, and rinsing or drying will cause changes in surface mass far surpassing the specific effect. In-situ measurement also offers the possibility of a one-step ELISA, in which the analyte, the conjugate and the chromogenic substrate are added together, without intervening washing or drying steps. In a normal ELISA this would be impossible, because the colour production by the excess of unbound conjugate would far surpass the colour production of the small amount of analyte-bound conjugate on the surface. In contrast, the presented technique only measures the precipitate produced by surface-bound conjugate. In addition to a higher sensitivity, the present use of in-situ measurement also allows a more rapid assay, because time-consuming rinsing and/or drying steps, with subsequent separate measurement of the precipitate, are avoided.
Another fundamental difference with the techniques described in the two mentioned patents is that no specially prepared optically active or polymer-coated surfaces are required according to the present invention. Such surfaces are expensive, just like the optically active slides used in techniques based on surface plasmon resonance (SPR), whereas cheap (disposable) reflecting surfaces, such as silicon wavers or chromium-sputtered glass slides, can be used in the present method.
The prior art suggests to use amplified ellipsometry as a means of enhancing the sensitivity of protein adsorption measurements [5]. In the prior art method concerned, the mass of the tagging antibody was increased by coupling it to silica particles. Antibody-coated gold particles have also been used, mainly combined with electron microscopy in localization studies [6]. Another prior art technique for amplification of surface mass uses biotinylated or analyte-coated liposomes [7,8].
These techniques are fundamentally different from the subject invention because they do not measure a continuing accumulation of precipitate per tagging molecule, but simply use heavier tags. Because of their slower diffusion towards the surface, the advantage of better detectability of such heavy tags has to be balanced against their slower adsorption, and this hampers the use of these methods for rapid assays.
The invention provides an immunoassay method for determining an analyte adsorbed at a surface or present in a liquid sample, comprising binding said analyte to a solid phase, attaching a marker to the analyte, and detecting marker attached to said solid-phase, wherein a combination of marker and detection means is used which is capable of producing a precipitate on a solid phase which carries the marker and binding of analyte to the solid phase is detected by in-situ determining surface mass of said solid phase due to formation of said precipitate.
Furthermore, the invention provides a kit for carrying out an immunoassay method for determining an analyte adsorbed at a surface or present in a liquid sample, wherein the kit comprises at least one means specifically adapted for carrying out the method according to the present invention.